Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-10-23
2004-10-12
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007930, C435S007940, C436S518000
Reexamination Certificate
active
06803202
ABSTRACT:
REFERENCE TO PRIOR APPLICATIONS
Reference is made to U.S. application Ser. No. 09/837,946, filed Apr. 19, 2001, entitled “Automated Computer Controlled Reporter Device for Conducting Immunoassay and Molecular Biology Procedures.”
FIELD OF THE INVENTION
This invention relates to detection of bioagents (and/or nucleic acids), and, more particularly, to a method for identifying the presence of bioagents and/or nucleic acids of specific interest in a specimen in the shortest possible time, with a minimum of necessary equipment and using known tests.
BACKGROUND
Health authorities are ever alert to the outbreak of a contagious disease that, if not checked early, could grow to epidemic proportion amongst the general population. To that purpose, the health authorities are charged with monitoring the environment for the incidence of unusual illnesses, symptomatic of a disease, obtain and analyze samples to identify the biological agent causing the disease, and, once identified, publish protective measures that must be taken to halt the spread of the disease. Typically, biological agents, such as virus and bacteria, are part of nature, and outbreaks of disease occur naturally. However, biological terrorism, the deliberate release of a harmful virus or bacteria or other biological agent (hereafter, “bioagent”) amongst a general population, and bio-warfare, the deliberate release of a bioagent against military troops in battle, are also of concern.
One known test procedure or process for detection of a specific bioagent that is applicable to a variety of fields, such as biotechnology, environmental protection and public health, is the enzyme linked immunoassay (hereafter referred to as “ELISA”). The ELISA process constitutes an identification process that uses molecular interactions to uniquely identify target substances. A basic definition of ELISA is a quantitative in vitro test for an antibody or antigen (e.g., a bioagent) in which the test material is adsorbed on a surface and exposed to a complex of an enzyme linked to an antibody specific for the substance being tested for with a positive result indicated by a treatment yielding a color in proportion to the amount of antigen or antibody in the test material. The basic ELISA procedure is described more specifically, for one, in a book entitled Methods in Molecular Biology Vol 42, John R. Crowther, Humana Press, 1995.
The “antibody specific for the substance being tested for” in the foregoing definition constitutes a recognition molecule, a molecule that is capable of binding to either reactant or product molecules in a structure-restricted manner. That is, the recognition molecule binds to a specific three-dimensional structure of a molecule or to a two-dimensional surface that is electrically charged and/or hydrophobic in a specific surface pattern. It may also be recognized that ELISA-like approaches using other recognition molecules can also be used, such as aptamers, DNA, RNA and molecular imprint polymers.
More recently, the foregoing definition for ELISA has been expanded beyond the colormetric approach, wherein color and color intensity is used as a reporter or indicia of the antigen or antibody, to include a voltametric or amperiometric approach to detection, wherein a rate of change of voltage or current conductivity in proportion to the amount of antigen or antibody contained in the test material. Patent Cooperation Treaty application PCT/US98/16714, filed Aug. 12, 1998 (International Publication No. WO 99/07870), entitled “Electrochemical Reporter System for Detecting Analytical Immunoassay and Molecular Biology Procedures” (hereafter the “16714 PCT application”), claiming priority of U.S. patent application Ser. Nos. 09/105,538 and 09/105,539”), to which the reader may refer, describes both a colormetric and an electrochemical reporter system for detecting and quantifying enzymes and other bioagents in analytical and clinical applications. The electrochemical reporter system of the 16714 PCT application employs a sensor for detecting voltametric and/or amperiometric signals that are produced in proportion to the concentration of organic (or inorganic) reporter molecules by redox (e.g. reduction-oxidation) recycling at the sensor.
In brief, in the ELISA test, the suspect bioagent is initially placed in a water-based buffer, such as a phosphate buffered saline solution, to form a sample solution. That sample solution is mixed with a quantity of particles, beads, the surface of which is coated with an antibody to the suspect bioagent, a recognition molecule (also sometimes referred to as a receptor molecule). The particular antibodies used to coat the beads are known to bind to the bioagent of interest or of concern and is a primary antibody or “1
o
Ab”. That is, the antibody coating exhibits a chemical “stickiness” that is selective to specific bioagents.
Any bioagent present in the sample solution binds with a non-covalent bond to a respective antibody and thereby becomes attached to a respective one of the beads in the mixture-solution. If the sample solution does not contain a bioagent or if the bioagent that is present in the solution is not one that binds to the selected antibody, then nothing binds to the foregoing antibody. Further processing of the ELISA process then shows nothing.
Assuming the suspect bioagent is present in the sample, the bioagent bonds to the antibody that is coated on the beads. The solution then contains a quantity of bioagent molecules bound respectively to a like quantity of coated beads. The mixture is optionally washed, as example, in a phosphate-buffered saline, and a second antibody, more specifically, an antibody and enzyme linked combination, is then added to the mixture. The second antibody is also one that is known to bind to the suspect bioagent, another recognition molecule. The second antibody may be either be one that is monoclonal, e.g. one that binds to only one specific molecule, or polyclonal, e.g. a mixture of different antibodies each of which shares the characteristic of bonding to the target bioagent. The enzyme, is covalently bound to the second antibody and forms a complex that is referred to as a secondary antibody-enzyme conjugate or “2
o
Ab-enz”. As known by those skilled in the art, an enzyme is a “molecule scissors”, a protein that catalyzes a biological reaction, a reaction that does not occur appreciably in the absence of the enzyme. The enzyme is selected to allow the subsequent production of an electrochemically active reporter.
The 2
o
Ab-enz binds to the exposed surface of the immobilized bioagent to form an “antibody sandwich” with the bioagent forming the middle layer of that sandwich. The antibody sandwich coated beads are washed again to wash away any excess 2
o
Ab-enz in the solution that remains unbound.
The beads and the attached antibody sandwich, the 1
o
Ab/bioagent/2
o
Ab-enz complex, in the solution are placed over the exposed surface of the redox recycling sensor. The substrate of the foregoing enzyme is added to the solution and the substrate is cleaved by the enzyme to produce an electrochemically active reporter. The substrate of the enzyme, referred to as PAP-GP, is any substance that reacts with an enzyme to modify the substrate. The effect of the enzyme is to separate, cut, the PAP, a para-amino phenol, the electrochemically active reporter, from the GP, an electrochemically inactive substance.
The foregoing chemical reaction is concentrated at the surface of the sensor. The rate of production of the foregoing reporter (PAP) is proportional to the initial concentration of bioagent. The reporter reacts at the surface of the sensor, producing an electrical current through the sensor that varies with time and is proportional to the concentration of the bioagent, referred to as redox recycling. The occurrence of the electric current constitutes a positive indication of the presence of the suspect bioagent in the sample. Analysis of the electric currents produced over an interval of time and comparison of the values of that electric current with
Sullivan Brian M.
Zsolnay Denes L.
Counts Gary W.
Goldman Ronald M.
Le Long V.
Northrop Grumman Corporation
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